JP7808832B2 - Composition for culture medium for feline mammary tumor organoids and method for producing feline mammary tumor organoids - Google Patents

Description

The present invention relates to a medium composition for culturing organoids prepared from feline mammary tumors and a method for producing feline mammary tumor organoids using the medium composition.

The three-dimensional organoid culture method (Non-Patent Document 1: Sato et al., Nature, 2009) was developed to reproduce three-dimensional epithelial tissue structures in culture dishes by mixing epithelial cells isolated from various organs with Matrigel and culturing them in a special medium containing factors that enhance stemness, such as Wnt, Noggin, and R-spondin. In recent years, organoid culture methods have been established using surgical specimens from human patients with colon cancer and pancreatic cancer, demonstrating structural similarity to tissue immediately after removal and correlation with genetic mutations (Non-Patent Document 2: Wetering et al., Cell, 2015 and Non-Patent Document 3: Boj et al., Cell, 2015), and is expected to become a useful tool for personalized medicine.

In addition, a technology has been developed to non-invasively cultivate bladder cancer organoids using urine samples from dogs with bladder cancer. It has been shown that the resulting organoids three-dimensionally reproduce the characteristics of bladder cancer in vivo and can be applied to tumor regeneration testing in immunodeficient mice and anticancer drug sensitivity testing for individual patients (Non-Patent Document 4: Elbadawy and Usui et al., Cancer Sci. 2019).

Mammary tumors are the third most common type of tumor in cats, accounting for approximately 17% of all tumors, and are known to be highly malignant with low survival rates. Mammary tumors also have a high recurrence rate, which means long-term treatment places a physical burden on patients and a financial burden on owners. Currently, surgery is the first choice of treatment for feline mammary tumors, with chemotherapy used in highly malignant cases, cases where surgery is not appropriate, and for palliative care. However, due to the high recurrence rate as mentioned above, anticancer drug therapy after surgical removal is recommended.

Anti-cancer drugs are often selected empirically by veterinarians, and no established treatment protocols exist. Meanwhile, there are no commercially available cultured cells for feline mammary tumors, so research leading to new treatments has stalled, making the establishment of effective anti-cancer drug treatments an urgent issue.

Sato et al., Nature, 459(7244):262-5, 2009 Wetering et al., Cell, 161(4):933-45, 2015 Boj et al., Cell, 160(1-2):324-38, 2015 Elbadawy and Usui et al., Cancer Sci. 110(9):2806-2821, 2019

The organoid culture method, which is currently attracting attention, uses a gel and a culture medium that stimulates stem cells and promotes their proliferation, allowing it to maintain cellular diversity and stemness while recreating the in vivo microenvironment. The use of cancer organoids obtained using this method in basic and clinical cancer research has attracted attention in recent years, but an organoid culture method for feline mammary tumors has not yet been established.

The present invention aims to produce feline mammary tumor organoids using mammary tumor tissue surgically removed from cats with mammary tumors, and to provide a medium composition optimal for culturing the feline mammary tumor organoids, a medium for the feline mammary tumor organoids, and a method for producing the feline mammary tumor organoids.

As a result of extensive research conducted by the inventors to achieve the above-mentioned objectives, they discovered that the proliferation rate of feline mammary tumor organoids is significantly improved when certain components are present during culture, leading to the completion of the present invention. The present invention encompasses the following:

[1] A composition for a feline mammary tumor organoid culture medium, comprising at least one component selected from the group consisting of FGF2, FGF7, FGF10, and TGF-α.
[2] The composition for a feline mammary tumor organoid culture medium according to [1], further comprising at least one component selected from the group consisting of a Wnt agonist, a BMP inhibitor, an EGF and a TGFβ inhibitor.
[3] The composition for a feline mammary tumor organoid culture medium according to [1], characterized in that the component is FGF10.
[4] A feline mammary tumor organoid culture medium containing at least one component selected from the group consisting of FGF2, FGF7, FGF10, and TGF-α.
[5] The cat mammary tumor organoid culture medium according to [4], further comprising at least one component selected from the group consisting of a Wnt agonist, a BMP inhibitor, an EGF and a TGFβ inhibitor.
[6] The cat mammary tumor organoid culture medium according to [4], characterized in that the component is FGF10.
[7] A method for producing feline mammary tumor organoids, comprising culturing feline mammary tumor organoids in a feline mammary tumor organoid medium described in any one of [4] to [6].
[8] The method for producing a feline mammary tumor organoid according to [7], further comprising the step of producing the feline mammary tumor organoid from mammary gland tissue collected from a cat suffering from mammary gland cancer.
[9] A step of preparing feline mammary tumor organoids from mammary glands collected from cats suffering from mammary gland cancer;
[4] to [6] A method for treating cats with mammary gland cancer, comprising the steps of culturing the feline mammary gland tumor organoids in the feline mammary gland tumor organoid culture medium described above, and further culturing the feline mammary gland tumor organoids obtained by the culture in the presence of a drug and measuring their drug sensitivity.
[10] The method of treatment described in [9], further comprising a step of administering to a cat with mammary gland cancer a drug that was found to have high drug sensitivity as a result of measuring the drug sensitivity.

The feline mammary tumor organoid culture medium composition of the present invention contains specific components, which can significantly improve the cell proliferation rate of feline mammary tumor organoids. Therefore, by using the feline mammary tumor organoid culture medium composition, feline mammary tumor organoids can be efficiently produced that can be used for basic research on feline mammary tumors and for treating cats with mammary tumors.

In addition, the method for producing feline mammary tumor organoids according to the present invention uses a medium containing specific components, allowing feline mammary tumor organoids to grow with excellent cell culture efficiency. Therefore, by using the method for producing feline mammary tumor organoids, it is possible to efficiently produce feline mammary tumor organoids that can be used for basic research on feline mammary tumors and for treating cats with mammary tumors.

Photographs of three types of feline mammary tumor organoids prepared in the examples. HE stained photographs of feline mammary tumor organoids and tumor tissue. Photographs of immunohistochemical staining showing the expression patterns of hormone receptors (HER2, ER, and PR) in feline mammary tumor organoids and tumor tissue. This is a photograph of feline mammary tumor organoids prepared in the examples when cultured in the presence of specific medium components. This is a characteristic diagram showing the results of measuring the cell proliferation efficiency when the feline mammary tumor organoids prepared in the examples were cultured in the presence of specific medium components. FIG. 1 is a characteristic diagram showing the results of an anticancer drug sensitivity test on the feline mammary tumor organoids prepared in the examples.

The present invention will be described in detail below.
The feline mammary tumor organoid culture medium composition of the present invention contains at least one component selected from the group consisting of FGF2, FGF7, FGF10, and TGF-α. Each of FGF2, FGF7, FGF10, and TGF-α can significantly improve the growth efficiency of feline mammary tumor organoids. That is, at least one component selected from the group consisting of FGF2, FGF7, FGF10, and TGF-α is used in a culture medium for culturing feline mammary tumor organoids. The feline mammary tumor organoids are not particularly limited and can be obtained by methods such as those described in Sato et al., Nature, 2009 and Sato T et al., Gastroenterology. 2011 Nov;141(5):1762-72. The feline mammary tumor organoid culture medium composition of the present invention is used in a culture medium for culturing feline mammary tumor organoids obtained by the method.

The feline mammary tumor organoid culture medium composition of the present invention may further contain some or all of the medium components for culturing feline mammary tumor organoids, which are described below. When the feline mammary tumor organoid culture medium composition of the present invention contains some of the medium components for culturing feline mammary tumor organoids, which are described below, it can be used as a medium for feline mammary tumor organoids together with the remaining medium components. Furthermore, when the feline mammary tumor organoid culture medium composition of the present invention contains all of the medium components for culturing feline mammary tumor organoids, which are described below, it can be used as a medium for feline mammary tumor organoids as is.

In the feline mammary tumor organoid culture medium composition of the present invention, the concentration of at least one component selected from the group consisting of FGF2, FGF7, FGF10, and TGF-α is not particularly limited and can be determined appropriately depending on the dilution ratio when used as a culture medium. Furthermore, when the feline mammary tumor organoid culture medium composition further contains some or all of the medium components used to culture feline mammary tumor organoids, the concentrations of these medium components are also not particularly limited and can be determined appropriately depending on the dilution ratio when used as a culture medium.

FGF2 is a basic fibroblast growth factor known as a non-glycosylated heparin-binding growth factor. There are no particular limitations on the FGF2, and FGF2 derived from any animal can be used. For example, commercially available FGF2 derived from various animals, such as human FGF2, mouse FGF2, or rat FGF2, can be used, or feline FGF2 produced as a recombinant by isolating the feline FGF2 gene can be used.

The concentration of FGF2 contained in the culture medium is not particularly limited, but can be, for example, 2 ng/mL to 500 ng/mL, preferably 5 ng/mL to 500 ng/mL, more preferably 5 ng/mL to 400 ng/mL, more preferably 5 ng/mL to 300 ng/mL, more preferably 5 ng/mL to 200 ng/mL, more preferably 5 ng/mL to 100 ng/mL, and even more preferably 5 ng/mL to 50 ng/mL. More specifically, the concentration of FGF2 contained in the culture medium can be 10 ng/mL.

FGF7 is a fibroblast growth factor, also known as keratinocyte growth factor (KGF). There are no particular limitations on the FGF7, and FGF7 derived from any animal can be used. For example, commercially available FGF7 derived from various animals, such as human FGF7, mouse FGF7, or rat FGF7, can be used, or feline FGF7 produced as a recombinant by isolating the feline FGF7 gene can be used.

The concentration of FGF7 contained in the culture medium is not particularly limited, but can be, for example, 0.4 ng/mL to 100 ng/mL, preferably 1 ng/mL to 100 ng/mL, more preferably 1 ng/mL to 80 ng/mL, more preferably 1 ng/mL to 60 ng/mL, more preferably 1 ng/mL to 40 ng/mL, more preferably 1 ng/mL to 20 ng/mL, and even more preferably 1 ng/mL to 10 ng/mL. More specifically, the concentration of FGF7 contained in the culture medium can be 5 ng/mL.

FGF10 is a fibroblast growth factor known as a heparin-binding growth factor. There are no particular limitations on the FGF10, and FGF10 derived from any animal can be used. For example, commercially available FGF10 derived from various animals, such as human FGF10, mouse FGF10, or rat FGF10, can be used, or feline FGF10 produced as a recombinant by isolating the feline FGF10 gene can be used.

The concentration of FGF10 contained in the culture medium is not particularly limited, but can be, for example, 4 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 1000 ng/mL, more preferably 10 ng/mL to 800 ng/mL, more preferably 10 ng/mL to 600 ng/mL, more preferably 10 ng/mL to 400 ng/mL, more preferably 10 ng/mL to 200 ng/mL, and even more preferably 10 ng/mL to 100 ng/mL. More specifically, the concentration of FGF10 contained in the culture medium can be 20 ng/mL.

TGF-α is a transforming growth factor-α (or transforming growth factor-α), a cytokine known to be produced by monocytes, keratinocytes, and various tumor cells. There are no particular limitations on the TGF-α, and TGF-α derived from any animal can be used. For example, commercially available TGF-α derived from various animals, such as human TGF-α, mouse TGF-α, or rat TGF-α, can be used, or feline TGF-α produced as a recombinant by isolating the feline TGF-α gene can be used.

The concentration of TGF-α contained in the culture medium is not particularly limited, but can be, for example, 4 ng/mL to 1000 ng/mL, preferably 10 ng/mL to 1000 ng/mL, more preferably 10 ng/mL to 800 ng/mL, more preferably 10 ng/mL to 600 ng/mL, more preferably 10 ng/mL to 400 ng/mL, more preferably 10 ng/mL to 200 ng/mL, and even more preferably 10 ng/mL to 100 ng/mL. More specifically, the concentration of TGF-α contained in the culture medium can be 20 ng/mL.

Culture components used with the feline mammary tumor organoid culture medium composition of the present invention include components contained in a typical culture medium for culturing three-dimensional organoids. Specifically, a typical culture medium for culturing three-dimensional organoids is not particularly limited, but a serum-free basal cell culture medium can be used. Examples of serum-free basal cell culture media include synthetic media with a pH of approximately 7.0 to 7.6 using a carbonate buffer. More specifically, Dulbecco's Modified Eagle Medium (Nutrient Mixture F-12; DMEM/F12) supplemented with glutamine, insulin, penicillin or streptomycin, and transferrin is included. Another example is Roswell Park Memorial Institute 1640 medium supplemented with glutamine, insulin, penicillin or streptomycin, and transferrin. Other examples include Advanced-DMEM/F12 supplemented with glutamine and penicillin or streptomycin, and Advanced RPMI medium supplemented with glutamine and penicillin or streptomycin.

The serum-free basal cell culture medium may also be supplemented with purified natural, semi-synthetic, or synthetic growth factors. Examples of growth factors include B-27 Supplement (Thermo Fisher Scientific), N-acetyl-L-cysteine (Sigma), and N-2 Supplement (Thermo Fisher Scientific).

In addition to these basic media, organoid culture media can also contain Wnt agonists that activate Wnt signaling, BMP inhibitors that inhibit BMP signaling, epidermal growth factor (EGF), and TGFβ inhibitors. Organoids are produced by culturing stem cells embedded in a scaffold support such as an extracellular matrix using these organoid production media. Examples of scaffold supports used in producing three-dimensional organoids include polymers that can absorb and retain water, such as collagen and agarose, and sponge-like membranes such as porous polystyrene. More specifically, Matrigel (manufactured by Corning Life Sciences), which contains collagen, can be used as a scaffold support.

Examples of Wnt agonists include Wnt, Wnt-3a, Noggin, GSK inhibitors, and R-spondins such as R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4.

BMP inhibitors include noggin, chordin, chordin-like proteins containing a chordin domain, follistatin, follistatin-related proteins containing a follistatin domain, DAN, DAN-like proteins containing a DAN cysteine-knot domain, sclerostin/SOST, decorin, and α-2 macroglobulin.

EGF is a 6045 Da protein consisting of 53 amino acid residues and three intramolecular disulfide bonds, and binds as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface. The concentration of EGF contained in the culture medium is not particularly limited, but can be, for example, 2 ng/mL to 500 ng/mL, preferably 5 ng/mL to 500 ng/mL, more preferably 10 ng/mL to 400 ng/mL, more preferably 20 ng/mL to 300 ng/mL, more preferably 30 ng/mL to 200 ng/mL, and even more preferably 40 ng/mL to 100 ng/mL. More specifically, the EGF concentration can be 50 ng/mL.

TGFβ (transforming growth factor β) inhibitors include, for example, A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate), SD-20 8 ((2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl-amine), SB-525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]quinoline), LY2157299 (4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxylic acid amide), TGF-β RI Kinase Inhibitor II 616452 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), TGF-β RI Kinase Inhibitor III 616453 (2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl), TGF-β RI Kinase Inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII Examples include 616458 (1-2-((6,7-dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone), naphthyridine (6-(2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)-quinoxaline), AP12009 (TGF-β2 antisense compound "Trabedersen"), Belagenpumatucel-L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human IgG4 monoclonal antibody that neutralizes TGF-β1)), and GC-1008 (anti-TGF-β monoclonal antibody).

The concentration of the TGF-β inhibitor contained in the culture medium varies depending on the type, and is not particularly limited. For example, when A83-01 is used as the TGF-β inhibitor, the concentration can be 0.1 μM to 5 μM, preferably 0.2 μM to 3 μM, more preferably 0.3 μM to 1 μM, and even more preferably 0.4 μM to 0.8 μM. More specifically, the concentration of A83-01 can be 0.5 μM.

Culture conditions for feline mammary tumor organoids are not particularly limited, and reference can be made, for example, to Sato et al., Nature, 2009 and Sato T et al., Gastroenterology. 2011 Nov;141(5):1762-72. When culturing feline mammary tumor organoids with reference to these publications, the culture temperature can be set to 30-40°C, with approximately 37°C being most preferable.

By using the feline mammary tumor organoid culture medium composition of the present invention, feline mammary tumor organoids can be cultured more efficiently than with conventional culture methods. In other words, by using the feline mammary tumor organoid culture medium composition of the present invention, the proliferation rate of feline mammary tumor organoids can be increased compared to conventional culture methods. In this way, by applying the present invention, the time required for subculture of feline mammary tumor organoids can be shortened, and they can be obtained more efficiently than when using conventional culture media.

Cultivated feline mammary tumor organoids can be used in a variety of bioassays, without particular limitations. For example, bioassays to confirm the effectiveness of anticancer drugs can be performed using cultured feline mammary tumor organoids. In particular, by applying the present invention, organoids can be produced from mammary tumors collected from cats with mammary tumors, and the cultured feline mammary tumor organoids can be used to verify the effectiveness (drug sensitivity) of anticancer drugs on the organoids, allowing for the appropriate selection of anticancer drugs that are effective in treating the cat. This allows for the rapid determination of the optimal treatment for cats with mammary tumors.

Furthermore, application of this invention makes it possible to rapidly supply feline mammary tumor organoids that can be used to develop new therapeutic drugs (anticancer drugs) for feline mammary tumors.

The present invention will be explained in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

Example 1
[Creation of feline mammary tumor organoids]
Three cats with mammary tumors underwent surgical resection of tumor sites. These tumors were designated FMT20001, FMT20002, and FMT20003. Feline mammary tumor organoids were then generated as follows: First, the harvested tissue was transferred to a 10 cm dish containing 2 ml of PBS to prevent drying. Using surgical scissors, tissue fragments approximately 1 cm square were then cut into pieces. The resulting tissue fragments were transferred to another 10 cm dish and washed three times with 2 ml of PBS to remove any blood components. The dish was then placed on ice and the tissue fragments were cut with ophthalmic scissors until they reached a viscous consistency. The tissue fragments were then transferred to a 15 ml tube containing 500 μl of Liberase TH 1.25 mg/ml (Sigma) and 450 μl of Advanced DMEM medium (Gibco) and pipetted 10 times using a 1 ml pipette. The tube was then placed in a 37°C incubator and shaken for 15 minutes. After 15 minutes, the tube was removed and pipetted 10 times using a 1ml pipetman, followed by another 15-minute shake. The tube was then removed and pipetted 10 times again to confirm that the cell suspension was translucent. The suspension was then passed through a 100µm cell strainer. If tissue fragments were still visible after 30 minutes of shaking, the tube was centrifuged at 600 × g for 3 minutes, the supernatant was removed, 1ml of TrypLE (Gibco) was added, and the suspension was placed in the incubator for 5 minutes. The tube was removed, pipetted as described above, and passed through a cell strainer. The filtered cell suspension was centrifuged at 600 × g for 3 minutes. After removing the supernatant, 8ml of PBS was added and pipetted 10 times using a 1ml pipetman. This procedure was repeated three times. The supernatant was removed, and 40 μl of Matrigel (Corning) was added per well of a 24-well plate, depending on the amount of cell pellet. The mixture was mixed gently several times using a 200 μl pipette. 40 μl of Matrigel containing the cell components was seeded into each well and placed in a 37°C incubator for 30 minutes. Then, 500 μl of medium preheated to 37°C was added per well to initiate the culture.

Using the above method, three types of feline mammary tumor organoids were generated (Figure 1). As shown in Figure 1, the three types of feline mammary tumor organoids generated in this study were observed to have different morphologies.

[Evaluation of homology between feline mammary tumor organoids and excised tissues]
HE staining of the feline mammary tumor organoids prepared as described above was performed according to standard methods. Furthermore, HE staining of the tumor tissues FMT20001 and FMT20002 excised as described above was also performed according to standard methods. HE staining images of the feline mammary tumor organoids and the original tumor tissues FMT20001 and FMT20002 are shown in Figure 2. As shown in Figure 2, the pathological structures of the feline mammary tumor organoids and the original tumor tissues were found to be very similar.

[Comparison of hormone receptor expression between feline mammary tumor organoids and tumor tissue]
The feline mammary tumor organoids prepared as described above and the original tumor tissue FMT20001 were evaluated for similarity in hormone receptor expression according to the following method. In this example, the expression of hormone receptors evaluated was HER2 (human epidermal receptor 2), ER (estrogen receptor), and PR (progesterone receptor).

First, frozen sections were prepared from the organoids and excised tissues. The slides were washed with PBS for 5 minutes with shaking. After removing from the PBS and wiping off the water, 50 μl of 1.5% normal goat serum (NGS) was added per section and allowed to stand at room temperature for 30 minutes in a humidified box. After removing the NGS, the primary antibody was diluted 1:100 with PBS and, as described above, 50 μl was added per section and allowed to stand overnight at 4°C. The next day, the slides were washed three times for 5 minutes each with PBS, and fluorescent secondary antibody and Hexst, each diluted in PBS, were applied at 50 μl per section and allowed to stand for 60 minutes in the dark. Subsequently, the slides were washed three times for 5 minutes each with PBS while protected from light, wiping off the water, mounted with a cover glass, and allowed to dry before being observed using a confocal laser scanning microscope (Zeiss).

The results of immunochemical staining experiments for HER2, ER, and PR in feline mammary tumor organoids and the original tumor tissue FMT20001 are shown in Figure 3. As shown in Figure 3, the expression patterns of HER2 and ER were found to be similar in feline mammary tumor organoids and the original tumor tissue FMT20001 (PR expression was not observed in either the organoids or tumor tissue).

[Investigation of optimal supplements for feline mammary tumor organoid culture]
In culturing the feline mammary tumor organoids prepared as described above, we searched for medium components that would further promote cell proliferation and organoid formation. Specifically, we prepared culture solutions with the medium composition shown in Table 1 to which various medium components were added, and compared the cell proliferation rate due to the addition of culture components, with the control set at 100%.

The medium components examined in this example are shown in Table 2.

Feline mammary tumor organoids (derived from tumor tissue FMT20001) were cultured for 7 days using culture medium supplemented with the medium components shown in Table 2. Images of the organoids after culture are shown in Figure 4, and the results of measuring cell proliferation rates are shown in Figure 5. The cell proliferation rate was calculated as follows: Culture medium was prepared by adding the various medium components shown in Table 2 to the medium composition shown in Table 1. Fluorescence intensity was measured using a plate reader (TECAN), and the cell proliferation rates of the other culture medium components were compared, with the control group containing only the medium composition shown in Table 1 set at 100%.

As shown in Figures 4 and 5, in addition to the well-known medium components used in the production of three-dimensional organoids, such as the Wnt agonist Wnt-3A and EGF, FGF2, FGF7, FGF10, and TGF-α had a proliferation-enhancing effect on feline mammary tumor organoids. In particular, FGF7 was found to have a superior proliferation-enhancing effect compared to Wnt-3A and EGF.

[Anticancer drug sensitivity test using feline mammary tumor organoids]
The feline mammary tumor organoids prepared in this study were found to be highly similar to the original feline mammary tumor tissue in terms of both structure and hormone receptor expression pattern. Therefore, we evaluated the responsiveness of the organoids to anticancer drugs, carboplatin and doxorubicin, by comparing their cell viability when used alone.

First, the medium was completely removed using an aspirator, and 500 μl of PBS was added per well. After aspirating the PBS, 500 μl of EDTA/PBS was added per well and the plate was left on ice for 30 minutes. The adhered Matrigel was then detached using a 1 ml pipette, and the plate was left on ice for another 60 minutes. The detached Matrigel was transferred to a 15 ml tube along with EDTA/PBS using a 1 ml pipette, and centrifuged at 600 × g for 3 minutes. The supernatant was removed using an aspirator, and 1 ml of PBS was added. The mixture was pipetted 10 times using a 1 ml pipette, and then centrifuged in the same manner. The supernatant was then removed, and 1 ml of TrypLE (Gibco) preheated to 37°C was added, pipetted 10 times, and the plate was left in a 37°C incubator for 5 minutes. The tube was removed and re-pipetted 10 times. A 70 μl cell strainer was attached to a 50 ml tube containing 100 μl of FBS, and the cell suspension was filtered. 10 μl of the prepared cell suspension was removed and used to calculate cell count. The amount of solution required for seeding cells into a 96-well plate was removed from the prepared cell suspension, transferred to a 1.5 ml tube, and centrifuged at 600 × g for 3 minutes. The supernatant was removed, and 10 μl of Matrigel was added per well of the 96-well plate. Mix gently several times using a 200 μl pipette. 10 μl of Matrigel containing cell components was seeded into each well and placed in a 37°C incubator for 30 minutes. 100 μl of prewarmed medium was then added per well and cultured. Carboplatin and doxorubicin were added the following day. After aspirating the medium, 100 μl of four concentrations of anticancer drug solution diluted with medium was added to each well. DMSO was used instead of the anticancer drug in the control group. Cell viability was measured three days later. 10 μl of PrestoBlue reagent (Invitrogen) was added to each well and the cells were incubated at 37°C. After 3-4 hours, fluorescence intensity was measured using a plate reader (TECAN) at Gain 45 to examine sensitivity to each anticancer drug.

The sensitivity of the three feline mammary tumor organoids (derived from FMT20001, FMT20002, and FMT20003) prepared above to carboplatin and doxorubicin is shown in Figure 6. As shown in Figure 6, it was clear that the three feline mammary tumor organoids used exhibited different sensitivities to carboplatin and doxorubicin. In particular, of the three feline mammary tumor organoids used, those derived from FMT20001 and FMT20003 showed extremely high sensitivity to carboplatin. These results suggest that carboplatin treatment is desirable for cats with mammary tumors from FMT20001 and FMT20003.

Claims (8)

A composition for a culture medium for feline mammary tumor organoids, comprising 10 ng/mL to 100 ng/mL of TGF-α. The feline mammary tumor organoid culture medium composition according to claim 1, further comprising at least one component selected from the group consisting of FGF2, FGF7, FGF10, a Wnt agonist, a BMP inhibitor, an EGF, and a TGFβ inhibitor. Feline mammary tumor organoid culture medium containing 10 ng/mL to 100 ng/mL of TGF-α. The feline mammary tumor organoid culture medium according to claim 3, further comprising at least one component selected from the group consisting of FGF2, FGF7, FGF10, a Wnt agonist, a BMP inhibitor, an EGF, and a TGFβ inhibitor. A method for producing feline mammary tumor organoids, comprising culturing feline mammary tumor organoids in the feline mammary tumor organoid medium described in claim 3 or 4. The method for producing feline mammary tumor organoids according to claim 5, further comprising the step of producing the feline mammary tumor organoids from mammary gland tissue collected from a cat with mammary gland cancer. A step of producing feline mammary tumor organoids from mammary glands collected from cats suffering from mammary gland cancer;
Culturing the feline mammary tumor organoid in the feline mammary tumor organoid culture medium according to claim 3 or 4;
Further culturing the feline mammary tumor organoids obtained by the culture in the presence of a drug and measuring drug sensitivity;
A method for treating cats with mammary gland cancer. The treatment method according to claim 7, further comprising the step of administering to a cat with mammary gland cancer a drug to which the drug sensitivity was determined to be high as a result of measuring the drug sensitivity.